Cdk inhibitors

ABSTRACT

The invention provides a compound represented by the following structural formula: (I) or a pharmaceutically acceptable salt, or a stereoisomer thereof useful for treating cancer.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of priority to International PatentApplication Number PCT/CN2020/088585, filed on May 5, 2020. The entirecontents of the aforementioned application are incorporated herein byreference.

BACKGROUND OF THE INVENTION

Cyclin-Dependent Kinases (CDKs) are a family of protein kinases firstdiscovered for their roles in regulating cell cycle. They have sincebeen identified to play roles in regulating a number of other biologicalfunctions such as transcription, mRNA processing, and thedifferentiation of nerve cells.

CDKs are relatively small proteins with molecular weights between about34-40 kDa. They contain little more than the kinase domain, and areessentially inactive when not in complex with a class of regulatoryproteins called cyclins. CDK levels remain relatively constantthroughout the cell cycle, and most regulation is post-translational,most prominently by binding to cyclins.

Like all kinases, the active site, or the ATP-binding site, of CDKs is acleft between a small amino-terminal lobe and a larger carboxy-terminallobe. The structure of human CDK2 revealed that CDKs have a modifiedATP-binding site that can be regulated by cyclin binding.Phosphorylation by CDK-activating kinase (CAK) at Thr 161 on the T-loopincreases the complex activity. Without cyclin, a flexible loop calledthe activation loop or T-loop blocks the cleft, and the position ofseveral key amino acid residues is not optimal for ATP-binding. Withcyclin, two alpha helices change position to permit ATP binding. One ofthem, the L12 helix that comes just before the T-loop in the primarysequence, becomes a beta strand and helps rearrange the T-loop, so it nolonger blocks the active site. The other alpha helix called the PSTAIREhelix rearranges and helps change the position of the key amino acidresidues in the active site.

Thus only the cyclin-CDK complex has active kinase activity, and most ofknown cyclin-CDK complexes regulate the progression through the cellcycle. The CDKs are ubiquitous in all known eukaryotes, and theirregulatory function in the cell cycle has been evolutionarily conserved.For example, yeast cells can proliferate normally when their CDK genehas been replaced with the homologous human gene. CDKs exert theirregulatory function by phosphorylating their substrates on certainspecific Serine and Threonine residues, and the consensus sequence of[S/T]PX[K/R], where S/T is the target Ser or Thr for phosphorylation, Pis proline, X is any amino acid, K is lysine, and R is arginine.

In animal cells, there are at least nine different CDKs, four of which(CDK1, 2, 3, and 4) are directly involved in cell cycle regulation. Inmammalian cells, CDK1, with its binding partners cyclin A2 and B1, alonecan drive the cell cycle. Cyclin-CDK complexes of earlier cell-cyclephase can help to activate cyclin-CDK complexes in later phase.

The same CDK may form complexes with different cyclins to regulatedifferent phases of the cell cycle. For example, CDK2 may form a complexwith cyclin D or E to regulate G1 phase; form a complex with cyclin A orE to regulate S phase; and form a complex with cyclin A to regulate G2phase. Meanwhile, CDK4 and CDK6 can form complexes with cyclins D1, D2,and D3.

The highly homologous Cyclin-dependent kinases (CDKs) CDK4 and CDK6 incombination with Cyclin D are key regulators of the transition throughthe restriction point R between the G1 (growth) and S (DNA replication)phases of the cell cycle. CDK4/6 exert their effects via phosphorylationof the retinoblastoma protein (pRb). Once phosphorylated, pRb loses itsinhibitory effect on the transcription of genes promoting entry into Sphase.

By contrast, specific inhibition of CDK4/6 kinase activity by theendogenous protein modulator p16^(INK4) or by small molecule inhibitorsresults in hypophosphorylated pRb and arrest of the cells at the G1restriction point. As the primary mechanism of regulating the G1restriction point, the pathway regulated by these kinases is altered ina broad spectrum of human tumors, and thus inhibition of CDK4/CDK6 inthese tumors has therapeutic benefit by preventing cell division.

There remains a need to provide CDK4/6 inhibitors which can be used inthe treatment of cell proliferative disorders such as cancer.

SUMMARY OF THE INVENTION

Described herein are compounds that inhibit the activity of acyclin-dependent kinase (CDK), e.g., CDK2, CDK4, and/or CDK6, andpharmaceutically acceptable salts, or stereoisomers thereof.

In one aspect, the invention provides a compound represented by thefollowing structural formula:

(Compound A), or a pharmaceutically acceptable salt thereof. It isdiscovered that Compound A not only actively inhibits CDK2, CDK4 andCDK6, but also demonstrates strong anti-proliferation activities.

In another aspect, the invention provides a compound represented by thefollowing structural formula:

or a pharmaceutically acceptable salt thereof. It is discovered thatCompound B selectively inhibit CDK4 but also has excellent brainpenetration.

Also provided are pharmaceutical compositions comprising the compoundsdisclosed herein, or a pharmaceutically acceptable salt, or astereoisomer thereof and a pharmaceutically acceptable carrier.

The present disclosure further provides a method of treating cancer in asubject in need thereof, comprising administering to the subject aneffective amount of (1) a compound disclosed herein or apharmaceutically acceptable salt, or a stereoisomer thereof; or (2) apharmaceutically acceptable composition comprising the compounddisclosed herein or a pharmaceutically acceptable salt, or astereoisomer thereof, and a pharmaceutically acceptable carrier. Incertain embodiments, the cancer is selected from the group consisting ofcolorectal cancer, breast cancer (such as hormone receptor positive,HER2/neu negative advanced or metastatic breast cancer in postmenopausalwomen), lung cancer, prostate cancer, glioblastoma, mantel celllymphoma, chronic myeloid leukemia and acute myeloid leukemia.

In certain embodiments of the methods of the invention, the cancer canbe treated by inhibiting the activity of a cyclin-dependent kinase(CDK), e.g., CDK2, CDK4, and/or CDK6.

In certain embodiments of the methods of the invention, the cancer iscarcinoma of the bladder, breast, colon, kidney, epidermis, liver, lung,oesophagus, gall bladder, ovary, pancreas, stomach, cervix, thyroid,nose, head and neck, prostate, or skin; a hematopoietic tumor oflymphoid lineage; a hematopoietic tumor of myeloid lineage; thyroidfollicular cancer; a tumor of mesenchymal origin; a tumor of the centralor peripheral nervous system; melanoma; seminoma; teratocarcinoma;osteosarcoma; xeroderma pigmentosum; keratoctanthoma; thyroid follicularcancer; or Kaposi's sarcoma.

In certain embodiments of the methods of the invention, the compoundsdisclosed herein are administered with any one of a second therapeuticagent as described herein that also treats the same cancer.

The present disclosure also provides a use of the compound disclosedherein or a pharmaceutically acceptable salt, or a stereoisomer thereofor a pharmaceutical composition comprising the same in any of themethods of the invention described above. In one embodiment, provided isthe compound disclosed herein or a pharmaceutically acceptable salt, ora stereoisomer thereof or a pharmaceutical composition comprising thesame for use in any of the method of the invention described above. Inanother embodiment, provided is use of the compound disclosed herein ora pharmaceutically acceptable salt, or a stereoisomer thereof or apharmaceutical composition comprising the same for the manufacture of amedicament for any of the method of the invention described.

DETAILED DESCRIPTION OF THE INVENTION 1. Overview

The present invention provides a compound of the present invention or apharmaceutically acceptable salt thereof for use in therapy, such ascancer therapy.

The present also invention provides a pharmaceutical formulationcomprising a compound of the present invention or a pharmaceuticallyacceptable salt thereof, and a pharmaceutically acceptable carrier,diluent, or excipient.

The present invention provides a compound of the present invention or apharmaceutically acceptable salt thereof for use in the treatment ofcancer. In particular, those cancers may be any of the cancers describedherein below, such as colorectal cancer, breast cancer (includingER⁺HER2⁻ advanced or metastatic or recurrent breast cancer is in anadult woman, or a postmenopausal woman), lung cancer, especiallynon-small cell lung cancer (NSCLC), prostate cancer, glioblastoma,mantel cell lymphoma (MCL), chronic myeloid leukemia (CML) and acutemyeloid leukemia (AML).

This invention further provides a method of treating cancer selectedfrom the group consisting of colorectal cancer, breast cancer (includingER⁺HER2⁻ advanced or metastatic or recurrent breast cancer is in anadult woman, or a postmenopausal woman), lung cancer, especiallynon-small cell lung cancer (NSCLC), prostate cancer, glioblastoma,mantel cell lymphoma, chronic myeloid leukemia and acute myeloidleukemia in a mammal comprising administering to a mammal in need ofsuch treatment an effective amount of a compound of the presentinvention or a pharmaceutically acceptable salt thereof.

Additionally, this invention provides the use of a compound of thepresent invention or a pharmaceutically acceptable salt thereof for themanufacture of a medicament for the treatment of cancer. In particularthose cancers are selected from the group consisting of colorectalcancer, breast cancer (including ER⁺HER2⁻ advanced or metastatic orrecurrent breast cancer is in an adult woman, or a postmenopausalwoman), lung cancer, especially non-small cell lung cancer (NSCLC),prostate cancer, glioblastoma, mantel cell lymphoma, chronic myeloidleukemia and acute myeloid leukemia.

Furthermore, this invention provides a pharmaceutical formulation foruse in therapy comprising a compound of the present invention or apharmaceutically acceptable salt thereof and a pharmaceuticallyacceptable carrier, diluent, or excipient. The invention also provides apharmaceutical formulation for treating colorectal cancer, breast cancer(including ER⁺HER2⁻ advanced or metastatic or recurrent breast cancer isin an adult woman, or a postmenopausal woman), lung cancer, especiallynon-small cell lung cancer (NSCLC), prostate cancer, glioblastoma,mantel cell lymphoma, chronic myeloid leukemia and acute myeloidleukemia comprising a compound of the present invention or apharmaceutically acceptable salt thereof and a pharmaceuticallyacceptable carrier, diluent, or excipient.

Treatable disease indications and potential second therapeutic agentuseful for combination therapy are described in further detail in thesections below.

It should be understood that any embodiment described herein, includingthose described only in one of the sections below or only in theexamples, may be combined with any one or more additional embodiments ofthe invention, unless expressly disclaimed or otherwiseimproper/inapplicable.

2. Definitions

Compounds described herein can comprise one or more asymmetric centers,and thus can exist in various stereoisomeric forms, e.g., enantiomersand/or diastereomers. For example, the compounds described herein can bein the form of an individual enantiomer, diastereomer or geometricisomer, or can be in the form of a mixture of stereoisomers, includingracemic mixtures and mixtures enriched in one or more stereoisomer.

Enantiomeric and diastereomeric mixtures can be resolved into theircomponent enantiomers or stereoisomers by well-known methods, such aschiral-phase gas chromatography, chiral-phase high performance liquidchromatography, crystallizing the compound as a chiral salt complex, orcrystallizing the compound in a chiral solvent. Enantiomers anddiastereomers can also be obtained from diastereomerically- orenantiomerically-pure intermediates, reagents, and catalysts bywell-known asymmetric synthetic methods. See, for example, Jacques etal., Enantiomers, Racemates and Resolutions (Wiley Interscience, NewYork, 1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, E. L.Stereochemistry of Carbon Compounds (McGraw-Hill, N Y, 1962); and Wilen,S. H. Tables of Resolving Agents and Optical Resolutions p. 268 (E. L.Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972).

When a compound is designated by a name or structure that indicates asingle enantiomer, unless indicated otherwise, the compound is at least60%, 70%, 80%, 90%, 99% or 99.9% optically pure (also referred to as“enantiomerically pure”). Optical purity is the weight in the mixture ofthe named or depicted enantiomer divided by the total weight in themixture of both enantiomers.

When the stereochemistry of a disclosed compound is named or depicted bystructure, and the named or depicted structure encompasses more than onestereoisomer (e.g., as in a diastereomeric pair), it is to be understoodthat one of the encompassed stereoisomers or any mixture of theencompassed stereoisomers are included. It is to be further understoodthat the stereoisomeric purity of the named or depicted stereoisomers atleast 60%, 70%, 80%, 90%, 99% or 99.9% by weight. The stereoisomericpurity in this case is determined by dividing the total weight in themixture of the stereoisomers encompassed by the name or structure by thetotal weight in the mixture of all of the stereoisomers.

When a geometric isomer is depicted by name or structure, it is to beunderstood that the geometric isomeric purity of the named or depictedgeometric isomer is at least 60%, 70%, 80%, 90%, 99% or 99.9% pure byweight. Geometric isomeric purity is determined by dividing the weightof the named or depicted geometric isomer in the mixture by the totalweight of both geometric isomers in the mixture.

Racemic mixture means 50% of one enantiomer and 50% of is correspondingenantiomer. The invention encompasses all enantiomerically-pure,enantiomerically-enriched, diastereomerically pure, diastereomericallyenriched, and racemic mixtures, and diastereomeric mixtures of thecompounds of the invention.

The compounds described herein may also include all isotopes of atomsoccurring in the intermediates or final compounds. Isotopes includethose atoms having the same atomic number but different mass numbers.For example, isotopes of hydrogen include tritium and deuterium.

It will be recognized that some variation of natural isotopic abundanceoccurs in a synthesized compound depending upon the origin of chemicalmaterials used in the synthesis. Thus, a preparation of the compounddisclosed herein will inherently contain small amounts of deuteratedisotopologues. The concentration of naturally abundant stable hydrogenand carbon isotopes, notwithstanding this variation, is small andimmaterial as compared to the degree of stable isotopic substitution ofcompounds of this invention. See, for instance, Wada, E et al.,Seikagaku, 1994, 66:15; Gannes, L Z et al., Comp Biochem Physiol MolIntegr Physiol, 1998, 119:725.

The compounds described herein may exist in various tautomeric forms.The term “tautomers” or “tautomeric” refers to two or moreinterconvertible compounds/substituents resulting from at least oneformal migration of a hydrogen atom and at least one change in valency(e.g., a single bond to a double bond, a triple bond to a single bond,or vice versa). Exemplary tautomerizations include keto-to-enol,amide-to-imide, lactam-to-lactim, enamine-to-imine, and enamine-to-(adifferent enamine) tautomerizations. The present teachings encompasscompounds in the form of tautomers, which includes forms not depictedstructurally. All such isomeric forms of such compounds are expresslyincluded. If a tautomer of a compound is aromatic, this compound isaromatic. Similarly, if a tautomer of a compound is a heteroaryl, thiscompound is heteroaryl.

In certain instances tautomeric forms of the disclosed compounds exist,such as the tautomeric structures shown below:

It is to be understood that when a compound herein is represented by astructural formula or designated by a chemical name herein, all othertautomeric forms which may exist for the compound are encompassed by thestructural formula.

The compounds of this invention can exist in free form for treatment, orwhere appropriate, as a pharmaceutically acceptable salt form.

The term “pharmaceutically acceptable salt” refers to those salts whichare, within the scope of sound medical judgment, suitable for use incontact with the tissues of humans and lower animals without unduetoxicity, irritation, allergic response, and the like, and arecommensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art, for example, Berge et al.describe pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein byreference. Pharmaceutically acceptable salts of the compounds of thisinvention include those derived from suitable inorganic and organicacids and bases. Examples of pharmaceutically acceptable, acid additionsalts are salts of an amino group formed with inorganic acids, such ashydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, andperchloric acid or with organic acids, such as acetic acid, oxalic acid,maleic acid, tartaric acid, citric acid, succinic acid, or malonic acidor by using other methods known in the art such as ion exchange. Otherpharmaceutically acceptable salts include adipate, alginate, ascorbate,aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate,camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate,glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate,hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts,and the like. Salts derived from appropriate bases include alkali metal,alkaline earth metal, ammonium, and N⁺(C₁₋₄ alkyl)₄ ⁻ salts.Representative alkali or alkaline earth metal salts include sodium,lithium, potassium, calcium, magnesium, and the like. Furtherpharmaceutically acceptable salts include, when appropriate, ammonium,quaternary ammonium, and amine cations formed using counterions such ashalide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkylsulfonate, and aryl sulfonate.

Such pharmaceutically acceptable acid addition salts and commonmethodology for preparing them are well known in the art. See, e.g.,Stahl et al., HANDBOOK OF PHARMACEUTICAL SALTS: PROPERTIES, SELECTIONAND USE, (VCHA/Wiley-VCH, 2002); Bighley et al., in “Encyclopedia ofPharmaceutical Technology.” Eds. Swarbrick and Boylan, Vol. 13, MarcelDekker, Inc., New York, Basel, Hong Kong 1995, pp. 453-499; Berge etal., “Pharmaceutical Salts,” Journal of Pharmaceutical Sciences, 66(1):1977.

The terms “composition” and “formulation” are used interchangeably.

A “subject” is a mammal, preferably a human, but can also be an animalin need of veterinary treatment, e.g., companion animals (e.g., dogs,cats, and the like), farm animals (e.g., cows, sheep, pigs, horses, andthe like) and laboratory animals (e.g., rats, mice, guinea pigs, and thelike).

The term “administer,” “administering,” or “administration” refers tomethods introducing a compound of the invention, or a compositionthereof, in or on a subject. These methods include, but are not limitedto, intraarticular (in the joints), intravenous, intramuscular,intratumoral, intradermal, intraperitoneal, subcutaneous, orally,topically, intrathecally, inhalationally, transdermally, rectally, andthe like. Administration techniques that can be employed with the agentsand methods described herein are found in e.g., Goodman and Gilman, ThePharmacological Basis of Therapeutics, current ed.; Pergamon; andRemington's, Pharmaceutical Sciences (current edition), Mack PublishingCo., Easton, Pa.

The terms “treatment,” “treat,” and “treating” refer to reversing,alleviating, or inhibiting the progress of a disease described herein.In some embodiments, treatment may be administered after one or moresigns or symptoms of the disease have developed or have been observed(i.e., therapeutic treatment). In other embodiments, treatment may beadministered in the absence of signs or symptoms of the disease. Forexample, treatment may be administered to a susceptible subject prior tothe onset of symptoms (i.e., prophylactic treatment) (e.g., in light ofa history of symptoms and/or in light of exposure to a pathogen).Treatment may also be continued after symptoms have resolved, forexample, to delay or prevent recurrence.

The terms “condition,” “disease,” and “disorder” are usedinterchangeably.

Generally, an effective amount of a compound taught herein variesdepending upon various factors, such as the given drug or compound, thepharmaceutical formulation, the route of administration, the type ofdisease or disorder, the identity of the subject or host being treated,and the like, but can nevertheless be routinely determined by oneskilled in the art. An effective amount of a compound of the presentteachings may be readily determined by one of ordinary skill by routinemethods known in the art.

The term “an effective amount” means an amount when administered to thesubject which results in beneficial or desired results, includingclinical results, e.g., inhibits, suppresses or reduces the symptoms ofthe condition being treated in the subject as compared to a control. Forexample, an effective amount can be given in unit dosage form (e.g.,from 1 mg to about 50 g per day, e.g., from 1 mg to about 5 grams perday).

A “therapeutically effective amount” is that amount effective fordetectable killing or inhibition of the growth or spread of cancercells; the size or number of tumors; or other measure of the level,stage, progression or severity of the cancer. The exact amount requiredwill vary from subject to subject, depending on the species, age, andgeneral condition of the subject, the severity of the disease, theparticular anticancer agent, its mode of administration, combinationtreatment with other therapies, and the like.

The general chemical terms used in the formulae above have their usualmeanings.

As used herein, “h” refers to hour or hours, “min” refers to minutes orminutes, “Cdk” or “CDK” refers to cyclin dependent kinase, “pRb” refersto retinoblastoma protein, “MCL” refers to mantle cell lymphoma, “AML”refers to acute myeloid leukemia, “CML” refers to chronic myeloidleukemia, “Boc” refers to N-tert-butoxycarbonyl, “EA” refers to ethylacetate, “DCM” refers to dichloromethane, “DMSO” refers todimethylsulfoxide, “DMA” refers to dimethylacetamide, “THF” refers totetrahydrofuran, “MtBE” refers to methyl tert-butyl ether, “TEA” refersto triethylamine, “FBS” refers to fetal bovine serum, “PBS” refers tophosphate buffered saline, “BSA” refers to bovine serum albumin, “RT”refers to room temperature, “mpk” means milligrams per kilogram, “po”refers to per os (oral), “qd” means once daily dosing, “HPLC” means highpressure liquid chromatography, “q2d” means a single dose every 2 days,“q2dx10” means a single dose every 2 days times 10, “VSMC” refers tovascular smooth muscle cell and “XRD” refers to X-ray diffraction.

3. Compounds

Another aspect of the present disclosure relates to labeled compounds ofthe invention (radio-labeled, fluorescent-labeled, etc.) that would beuseful not only in imaging techniques but also in assays, both in vitroand in vivo, for localizing and quantitating CDK in tissue samples,including human, and for identifying CDK ligands by inhibition bindingof a labeled compound. Accordingly, the present disclosure includes suchlabeled compounds.

The present disclosure further includes isotopically-labeled compoundsof the invention. An “isotopically” or “radio-labeled” compound is acompound of the invention where one or more atoms are replaced orsubstituted by an atom having an atomic mass or mass number differentfrom the atomic mass or mass number typically found in nature (i.e.,naturally occurring). Suitable radionuclides that may be incorporated incompounds of the present invention include but are not limited to ²H(also written as D for deuterium), ³H (also written as T for tritium),¹¹C, ¹³C, ¹⁴C, ¹³N, ¹⁵N, ¹⁵O, ¹⁷O, ¹⁸O, ¹⁸F, ³⁵S, ³⁶Cl, ⁸²Br, ⁷⁵Br,⁷⁶Br, ⁷⁷Br, ¹²³I, ¹²⁴I, ¹²⁵I and ¹³¹I. The radionuclide that isincorporated in the instant radio-labeled compounds will depend on thespecific application of that radio-labeled compound.

The present invention can further include synthetic methods forincorporating radio-isotopes into compounds of the invention. Syntheticmethods for incorporating radio-isotopes into organic compounds are wellknown in the art, and an ordinary skill in the art will readilyrecognize the methods applicable for the compounds of invention.

A labeled compound of the invention can be used in a screening assay toidentify/evaluate compounds. For example, a newly synthesized oridentified compound (i.e., test compound) which is labeled can beevaluated for its ability to bind a CDK by monitoring its concentrationvariation when contacting with the CDK, through tracking of thelabeling. For example, a test compound (labeled) can be evaluated forits ability to reduce binding of another compound which is known to bindto a CDK (i.e., standard compound). Accordingly, the ability of a testcompound to compete with the standard compound for binding to the CDKdirectly correlates to its binding affinity. Conversely, in some otherscreening assays, the standard compound is labeled and test compoundsare unlabeled. Accordingly, the concentration of the labeled standardcompound is monitored in order to evaluate the competition between thestandard compound and the test compound, and the relative bindingaffinity of the test compound is thus ascertained.

In one embodiment, the compound or a pharmaceutically acceptable salt,or a stereoisomer thereof, wherein one or more hydrogen atoms arereplaced by deuterium.

4. Treatable Diseases and Method of Treatment

Certain compounds of the present invention are selective inhibitors ofCDK2, CDK4, and/or CDK6, and are therefore useful in the treatment of adisease or disorder characterised by abnormal cell proliferation thatcan be inhibited by a reduced activity of CDK-cyclin complexesencompassing CDK2, CDK4, and/or CDK6.

In certain embodiments, compounds of the invention selectively inhibitCDK4/6 over CDK2, with a ratio of IC₅₀ values for the latter (CDK2)against the former (CDK4/6) of at least about 10, 20, 50, 100, 200, 300,400, 500, 800, 1,000, 2,000 or more.

In certain embodiments, compounds of the invention selectively inhibitCDK4 over CDK6, with a ratio of IC₅₀ values for the latter (CDK6)against the former (CDK4) of at least about 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, 50 or more.

In certain embodiments, compounds of the invention selectively inhibitCDK2 over CDK4, with a ratio of IC₅₀ values for the latter (CDK4)against the former (CDK2) of at least about 2, 5, 10, 15, 20, 40, 50,60, 80, 100 or more.

In certain embodiments, compounds of the invention inhibits CDK2/4/6with similar IC₅₀ values, e.g., IC₅₀ values within 10-, 5-, 3-, or2-fold. Such compounds of the invention are useful for treating cancerswith cyclin D1 or E1 or E2 amplification or enhanced expression.

CDK2 is the catalytic subunit of the CDK-cyclin complex whose activityis restricted to the G1-S phase of the cell cycle, where cells makeproteins necessary for mitosis and replicate their DNA. CDK2 iscomplexed with cyclin E or A. Cyclin E binds G1 phase CDK2, which isrequired for G1 to S phase transition. On the other hand, CDK2 bindingwith Cyclin A is required to progress through the S phase.

Although CDK2 is mostly dispensable in the cell cycle of normallyfunctioning cells, it is critical to the abnormal growth processes ofcancer cells. Overexpression of cyclin E occurs in many tumor cells,causing the cells to become dependent on CDK2 and cyclin E. Abnormalcyclin E activity is observed in breast, lung, colorectal, gastric, andbone cancers, as well as in leukemia and lymphoma. Likewise, abnormalexpression of cyclin A2 is associated with chromosomal instability andtumor proliferation, while inhibition leads to decreased tumor growth.Therefore, CDK2 and its cyclin binding partners represent possibletherapeutic targets for new cancer therapeutics. Pre-clinical modelshave shown preliminary success in limiting tumor growth, and have alsobeen observed to reduce side effects of current chemotherapy drugs.

For example, Caldon et al. (Mol Cancer Ther 11(7):1488-1499, 2012)reported that Cyclin E2 is included in several gene signatures thatpredict disease progression in either tamoxifen-resistant or metastaticbreast cancer, and high expression of CycE2 was characteristic of theluminal B and HER2 subtypes of breast cancer and was strongly predictiveof shorter distant metastasis-free survival following endocrine therapy.Further, tamoxifen-resistant (MCF-7 TAMR) breast cancer cellsoverexpressed cyclin E2; and expression of either cyclin E1 or E2 inT-47D breast cancer cells conferred acute antiestrogen resistance,suggesting that cyclin E overexpression contributes to the antiestrogenresistance of tamoxifen-resistant cells. Proliferation oftamoxifen-resistant cells was inhibited by RNAi-mediated knockdown ofcyclin E1, cyclin E2, or CDK2. Besides, ectopic expression of cyclin E1or E2 also reduced sensitivity to CDK4, but not CDK2, inhibition.Furthermore, CDK2 inhibition of E-cyclin overexpressing cells andtamoxifen-resistant cells restored sensitivity to tamoxifen or CDK4inhibition.

These data demonstrate that Cyclin E2 overexpression is a potentialmechanism of resistance to both endocrine therapy and CDK4 inhibition,and CDK2 inhibitors may in turn overcome such resistance, and may bebeneficial as a component of combination therapies inendocrine-resistant disease as they effectively inhibit cyclin E1 and E2overexpressing cells and enhance the efficacy of other therapeutics.Likewise, the subject compounds with potent inhibitory activitiesagainst both CDK2 and CDK4 are expected to be effective against cancercells that are both non-resistant and resistant to endocrine therapy orCDK4 inhibition.

Thus in certain embodiments, the compounds of the invention may havepotent inhibitory effects against both CDK2 and CDK4 (e.g.,independently <10 nM, <5 nM, <1 nM level of IC₅₀ values), and thus areeffective to treat tamoxifen-resistant or metastatic breast cancers,such as tamoxifen-resistant or metastatic breast cancers with CycEoverexpression.

IC₅₀ values of the compounds of the invention against CDK2/4/6 can bemeasured using, for example, the methods described in Examples 1-3(incorporated herein by reference).

In particular, the compounds of the present invention are useful in thetreatment of cancer. In other embodiments, the compounds of the presentinvention are useful in the treatment of chronic inflammation diseasessuch as arthritis and cystic fibrosis.

Thus in one aspect, the present invention provides a method of treatingcancer, in particular the cancers described herein, in a mammal,comprising administering to a mammal in need of such treatment aneffective amount of a compound of the present invention.

In a related aspect, the invention is directed to a use of a compound ofthe present invention in the manufacture of a medicament for treatingcancer, in particular, the cancers described herein.

In another related aspect, the compounds of the present invention can beused in the manufacture of a medicament for the treatment of cancer, inparticular, the cancers described herein.

In another related aspect, the invention provides a compound of thepresent invention for use in treating cancer, in particular, the cancersdescribed herein.

According to any of the above related aspects of the invention, CDK4 andCDK6 may modulate their effects on the cell cycle partly through pRbphosphorylation. Thus, certain compounds of the present invention mayinhibit pRb phosphorylation through inhibiting CDK4/6 activity, and thusinhibiting cell proliferation and/or tumor growth, in any cancer typewhere the cells are proliferating and contain a functional, intact Rb1gene that encodes pRb.

Thus in certain embodiments, the compounds of the invention are usefulin the treatment of pRb⁺ cancers, such as colorectal cancer, breastcancer, lung cancer, prostate cancer, chronic myeloid leukemia, acutemyeloid leukemia (Fry et al., Mol. Cancer Ther. 3(11):1427, 2004),mantel cell lymphoma (Marzec et al., Blood 108(5):1744, 2006), ovariancancer (Kim et al., Cancer Research 54:605, 1994), pancreatic cancer(Schutte et al., Cancer Research 57:3126, 1997), malignant melanoma andmetastatic malignant melanoma (Maelandsmo et al., British Journal ofCancer 73:909, 1996) in mammals. The compounds of the invention are alsoexpected to be useful in the treatment of rhabdomyosarcoma (Saab et al.,Mol. Cancer. Ther. 5(5):1299, 2006) and multiple myeloma (Baughn et al.,Cancer Res. 66(15):7661, 2006), including relapsed refractory multiplemyeloma, in mammals (e.g., human).

Meanwhile, Zhang et al. (Nature dx.doi.org/10.1038/nature25015, 2017)reported that inhibition of CDK4/6 in vivo may lead to decreasedphosphorylation and therefore increased degradation of Cullin 3^(SPOP)E3 ligase (by APC/C^(Cdh1)), which in turn leads to increased PD-L1levels on tumor cell surface, and reduced numbers of tumor-infiltratinglymphocytes (TILs) in mouse tumors and in primary human prostate cancerspecimens. In other words, inhibition of CDK4/6 in vivo elevates PD-L1protein levels, and contributes to increased resistance to immunecheckpoint therapy targeting PD-1 (programmed cell death protein 1) andPD-L1 (ligand for PD-1). On the other hand, combining CDK4/6 inhibitortreatment with anti-PD-1 immunotherapy enhances tumor regression, anddramatically improves overall survival rates in mouse tumor models.

Thus in certain embodiments, the compounds of the invention can be usedin combination with PD-1/PD-L1 immune checkpoint inhibitors to enhancetherapeutic efficacy for human cancers.

PD-1 and PD-L1 inhibitors that can be used with the compounds of theinvention are known in the art. PD-1 inhibitors include monoclonalantibodies or antigen binding fragment thereof specific for PD-1.Exemplary PD-1 inhibitors include Pembrolizumab (Keytruda), Nivolumab(Opdivo), and Cemiplimab (Libtayo). PD-L1 inhibitors include monoclonalantibodies or antigen binding fragment thereof specific for PD-L1.Exemplary PD-L1 inhibitors include Atezolizumab (Tecentriq), Avelumab(Bavencio), and Durvalumab (Imfinzi).

Additional immune checkpoint inhibitor that may be used with thecompounds of the invention for enhancing therapeutic efficacy for humancancers include monoclonal antibodies or antigen binding fragmentsthereof specific for CTLA-4 such as Ipilimumab (Yervoy).

Further immune checkpoint inhibitor that may be used with the compoundsof the invention for enhancing therapeutic efficacy for human cancersinclude bispecific monoclonal antibodies or antigen binding fragmentsthereof specific for PD-1 and PD-L1, or combination of monoclonalantibodies or antigen binding fragments thereof specific for PD-1 andPD-L1, or PD-1 and CTLA-4, etc.

In certain embodiments, the compounds of the invention can be used incombination with Tyr kinase inhibitor, e.g., receptor Tyr kinase (RTK)inhibitors, to enhance therapeutic efficacy for human cancers. ExemplaryTyr kinase inhibitors include ALK inhibitors (such as Crizotinib,Ceritinib, Alectinib, Brigatinib), Bcr-Abl inhibitors (such asBosutinib, Dasatinib, Imatinib, Nilotinib, Ponatinib), BTK inhibitor(such as Ibrutinib), c-Met inhibitor (such as Crizotinib, Cabozantinib),EGFR inhibitor (such as Gefitinib, Erlotinib, Lapatinib, Vandetanib,Afatinib, Osimertinib), JAK inhibitor (such as Ruxolitinib,Tofacitinib), MEK1/2 inhibitor (such as Trametinib), PDGFR inhibitor(such as Axitinib, Gefitinib, Imatinib, Lenvatinib, Nintedanib,Pazopanib, Regorafenib, Sorafenib, Sunitinib), RET inhibitor (such asVandetanib), Src family kinase inhibitors (such as Bosutinib, Dasatinib,Ponatinib, Vandetanib), and VEGFR family inhibitors (such as Axitinib,Lenvatinib, Nintedanib, Regorafenib, Pazopanib, Sorafenib, Sunitinib).

Additional suitable kinase inhibitors that can be used in combinationwith the subject compounds, as well as the treatable cancer indications,are described in Bhullar et al., Molecular Cancer 17:48, 2018(incorporated herein by reference in its entirety).

Further additional RTK inhibitors include monoclonal antibodies andantigen-binding fragments thereof, including the anti-EGFR mAB such ascetuximab (effective in treating, e.g., lung, colorectal, and head andneck cancer), and the anti-HER2 mAb such as trastuzumab (effective totreat, for example, breast cancer).

In certain embodiments, the compounds of the invention can be used incombination with an antagonist of hormonal receptor signaling, such asthe ones described before for breast cancer treatment.

Cancers treatable with the compounds of the invention include:Non-Hodgkin's lymphoma; malignant mesothelioma; non-small cell lungcancer; cholangiocarcinoma; soft tissue sarcoma; glioblastoma;(recurrent) brain tumor; brain metastases secondary to hormone receptorpositive breast cancer, non-small cell lung cancer, melanoma (includingmelanoma positive for cyclin D1 expression); (recurrent or persistent)endometrial cancer; (recurrent or metastatic) Head and Neck SquamousCell Carcinoma (HNSCC); hepatocellular carcinoma; esophageal squamouscell carcinoma (SCC); esophageal adenocarcinoma (ADC); renal cellcarcinoma, and urothelial cancer.

In certain embodiments, the treatable cancers include: carcinoma of thebladder, breast, colon, kidney, epidermis, liver, lung (including SCLCand NSCLC), esophagus, gall bladder, ovary, pancreas, stomach, cervix,thyroid, nose, head and neck, prostate, or skin; a hematopoietic tumorof lymphoid lineage; a hematopoietic tumor of myeloid lineage; thyroidfollicular cancer; a tumor of mesenchymal origin; a tumor of the centralor peripheral nervous system; melanoma; familial melanoma; seminoma;teratocarcinoma; osteosarcoma; xeroderma pigmentosum; keratoctanthoma;thyroid follicular cancer; Kaposi's sarcoma, squamous cancer, sarcoma;or a tumor of mesenchymal origin.

In certain embodiments, the hematopoietic tumor of lymphoid lineage isleukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia,B-cell lymphoma, T-cell lymphoma, multiple myeloma, Hodgkin's lymphoma,non-Hodgkin's lymphoma, hairy cell lymphoma, or Burkett's lymphoma.

In certain embodiments, the tumor of the central or peripheral nervoussystem is astrocytoma, neuroblastoma, glioma or schwannoma.

In certain embodiments, the cancer is small cell lung cancer, non-smallcell lung cancer, pancreatic cancer, breast cancer, glioblastomamultiforme, T cell ALL and mantle cell lymphoma.

In certain embodiments, the cancer is selected from the group consistingof: colorectal cancer, mantel cell lymphoma, breast cancer (includingadvanced or metastatic or recurrent breast cancer), pancreatic cancer,ovarian cancer, glioblastoma, acute myeloid leukemia, and lung cancer,especially NSCLC.

In certain embodiments, the cancer is NSCLC, pancreatic cancer, ovariancancer or metastatic breast cancer, and the treatment comprisingadministering to a mammal in need thereof a therapeutically effectivecombination of a compound of the present invention and gemcitabine HCl.

In certain embodiments, the cancer is NSCLC, pancreatic cancer, ovariancancer or metastatic breast cancer, wherein the medicament comprisingthe compound of the present invention also comprises gemcitabine HCl, oris to be administered simultaneously, separately or sequentially withgemcitabine HCl.

In certain embodiments, the compounds of the present invention can beused in combination with other agents for the treatment of NSCLC,pancreatic cancer, ovarian cancer and metastatic breast cancer. Forexample, the compound of the present invention may be used insimultaneous, separate or sequential combination with gemcitabine HCl inthe treatment of NSCLC, pancreatic cancer, ovarian cancer or metastaticbreast cancer.

In certain embodiments, the cancer is selected from the group consistingof colorectal cancer, glioblastoma, acute myeloid leukemia and lungcancer.

In certain embodiments, the cancer is glioblastoma or astrocytoma, andthe treatment utilizes a therapeutically effective combination of acompound of the invention and temozolomide. The compound of theinvention may be administered simultaneously, separately or sequentiallywith temozolomide.

Breast Cancer Treatment

In certain embodiments, the compounds of the invention can be used totreat breast cancer.

Breast cancer presents a significant health burden worldwide, and italone accounted for ˜7% of all US cancer-related deaths in 2016. Of allbreast cancers, about 75% are diagnosed as hormone receptor-positive(HR⁺) breast cancer, which expresses the estrogen receptor (ER) and/orthe progesterone receptor (PgR), and is typically dependent on the ERsignaling pathway for growth and survival. That is, the HR⁺ breastcancers harness the biological functions of the ER pathway to promotebreast cancer growth, development, and progression. Meanwhile, thereliance of HR⁺ breast cancer on ER signaling made such breast cancer atherapeutic target for endocrine therapy agents that target the estrogensignaling pathway, such as aromatase inhibitors (AIs; includingletrozole, anastrozole, and exemestane), selective ER modulators(tamoxifen), and selective ER down-regulators (fulvestrant), etc.

Although endocrine therapy makes up the treatment backbone for HR⁺breast cancer, the efficacy of endocrine therapy is limited by highrates of both pre-existing de novo resistance, and resistance acquiredduring treatment, due to the presence of alternative survival or“escape” pathway. The ER pathway and many of the known escape pathwaysact through the cyclin D-CDK4/6-inhibitor of CDK4 (INK4)-retinoblastoma(Rb) pathway to promote tumor growth. As such, targeting both the ER andthe cyclin D-CDK4/6-INK4-Rb pathways in combination usually lead to amore extensive inhibition of tumor growth and prevent the activation ofescape pathways, precluding the development of endocrine therapyresistance. See Sammons et al., Current Cancer Drug Targets 17:637-649,2017.

Thus in certain embodiments, the breast cancer is a pRb+ breast cancer.In certain embodiments, the breast cancer is a hormone receptor(HR)-positive (e.g., estrogen receptor positive (ER⁺), progesteronereceptor positive (PR⁺), or ER⁺PR⁺), HER2/neu-negative cancer, includingHR⁺HER2⁻ or ER⁺HER2⁻, advanced or metastatic or recurrent breast cancer.In certain embodiments, the HR⁺HER2⁻ or ER⁺HER2⁻ advanced or metastaticor recurrent breast cancer is in an adult woman, or a postmenopausalwoman.

In certain embodiments, the compounds of the invention is either usedalone, or used with an aromatase inhibitor (that inhibits estrogenproduction), to treat HR-positive, HER2-negative advanced or metastaticor recurrent breast cancer. In certain embodiments, the aromataseinhibitor temporarily inactivate aromatase (such as anastrozole(ARIMIDEX®) and letrozole (FEMARA®)). In certain embodiments, thearomatase inhibitor permanently inactivate aromatase (such as exemestane(AROMASIN®)).

In certain embodiments, the compound(s) of the invention is used with acompound that interferes with estrogen's ability to stimulate the growthof breast cancer cells, such as a Selective Estrogen Receptor Modulator(SERM) that binds to the estrogen receptor to prevent estrogen binding,such as tamoxifen (NOLVADEX®) and toremifene (FARESTON®). Tamoxifen hasbeen used for more than 30 years to treat HR⁺ breast cancer.

In certain embodiments, the compound(s) of the invention is used with apure antiestrogen with no estrogen agonist activity, such as fulvestrant(FASLODEX®).

In certain embodiments, the HR-positive, HER2-negative advanced ormetastatic or recurrent breast cancer is in a postmenopausal woman. Incertain embodiments, the HR-positive, HER2-negative advanced ormetastatic or recurrent breast cancer has progressed after takingtherapy that alters a patient's hormones (e.g., estrogen and/orprogesterone), or has worsened after treatment with another hormonetherapy.

In certain embodiments, the compound(s) of the invention is used in apatient undergoing ovarian ablation, or has received ovarian ablation.In certain embodiments, the ovarian ablation is through oophorectomy orradiation treatment.

In certain embodiments, the compound(s) of the invention is used with acompound that temporarily suppresses ovarian function (e.g., estrogenand/or progesterone production). Such compound includesgonadotropin-releasing hormone (GnRH) agonists or luteinizinghormone-releasing hormone (LH-RH) agonists, including goserelin(ZOLADEX®) and leuprolide (LUPRON®).

In certain embodiments, the compound(s) of the invention is used with acompound that inhibits CYP3A4, such as ritonavir, indinavir, nelfinavir,saquinavir, clarithromycin, telithromycin, chloramphenicol,ketoconazole, itraconazole, posaconazole, voriconazole, nefazodone,cobicistat, amiodarone, aprepitant, verapamil, diltiazem, erythromycin,fluconazole, miconazole, bergamottin, cimetidine, ciprofloxacin,cyclosporine, donedarone, fluvoxamine, imatinib, Valerian,buprenorphine, cafestol, cilostazol, fosaprepitant, gabapentin,lomitapide, orphenadrine, ranitidine, ranolazine, tacrolimus,ticagrelor, valproic acid, amlodipine, cannabidiol, dithiocarbamate,mifepristone, norfloxacin, delavirdine, gestodene, mibefradil, starfruit, milk thistle, niacinamide, Ginkgo biloba, piperine, isoniazid,and quercetin.

In certain embodiments, the compound(s) of the invention is used with aninhibitor of IGF-1/IGF-2, such as a monoclonal antibody or anantigen-binding fragment thereof against IGF-1/IGF-2. Exemplaryantibodies include xentuzumab, a humanized IgG1 mAb.

In certain embodiments, the compound(s) of the invention is used with acompound that inhibits PI3K. It is believed that inhibition of PI3Kreduces the levels of cyclin D1 and other G1-S cyclins, abolishes pRbphosphorylation, and inhibits activation of S-phase transcriptionalprograms. Representative PI3K inhibitors for use with the compounds ofthe invention includes idelalisib, copanlisib, duvelisib, taselisib,perifosine, buparlisib, alpelisib, umbralisib, copanlisib, dactolisib,and voxtalisib.

In certain embodiments, the mammal to be treated is a human, such as anadult woman having breast cancer (e.g., postmanupausal woman or adultwoman having hormone receptor (HR)-positive, human epidermal growthfactor receptor 2 (HER2)-negative advanced or metastatic or recurrentbreast cancer that has progressed after taking therapy that alters apatient's hormones).

Additionally, certain compounds of the present invention exhibit theadvantageous property that they are able to cross the blood-brainbarrier. Such compounds are therefore able to penetrate the brain andare thus useful in the treatment of primary and metastatic brain tumorswhere the cells are proliferating and contain a functional, intact Rb1gene. Examples of such pRb⁺ brain tumors include glioblastoma, as wellas medulloblastoma and astrocytoma (Lee et al., Science 235:1394, 1987).

Temozolomide is a cytotoxic, DNA alkylating agent used for the treatmentof brain tumors including glioblastoma and astrocytoma (Friedman et al.,Clin. Cancer Res. 6(7):2585-2597, 2000) including brain metastases frommelanoma, breast cancer and NSCLC (Siena et al., Annals of Oncology,doi:10.1093/annonc/mdp343, 2009). Temozolomide interacts with DNAcausing chemical modification/damage (Marchesi et al., Pharmacol. Res.56(4):275-287, 2007). Thus, in some embodiments, the compounds of thepresent invention can be used in combination with temozolomide for thetreatment of primary and metastatic pRb⁺ brain tumors such asglioblastoma and astrocytoma, for example, where such metastases arederived from melanoma, breast cancer or NSCLC.

5. Pharmaceutical Compositions

The invention provides pharmaceutical compositions which comprise anyone of the compounds described herein, or a pharmaceutically acceptablesalt thereof, and one or more pharmaceutically acceptable carriers orexcipients.

“Pharmaceutically acceptable excipient” and “pharmaceutically acceptablecarrier” refer to a substance that aids the formulation and/oradministration of an active agent to and/or absorption by a subject andcan be included in the compositions of the present disclosure withoutcausing a significant adverse toxicological effect on the subject.Non-limiting examples of pharmaceutically acceptable carriers andexcipients include water, NaCl, normal saline solutions, lactatedRinger's, normal sucrose, normal glucose, binders, fillers,disintegrants, lubricants, coatings, sweeteners, flavors, salt solutions(such as Ringer's solution), alcohols, oils, gelatins, carbohydratessuch as lactose, amylose or starch, fatty acid esters,hydroxymethycellulose, polyvinyl pyrrolidine, and colors, and the like.Such preparations can be sterilized and, if desired, mixed withauxiliary agents such as lubricants, preservatives, stabilizers, wettingagents, emulsifiers, salts for influencing osmotic pressure, buffers,coloring, and/or aromatic substances and the like that do notdeleteriously react with or interfere with the activity of the compoundsprovided herein. One of ordinary skill in the art will recognize thatother pharmaceutical carriers and excipients are suitable for use withdisclosed compounds.

These compositions optionally further comprise one or more additionaltherapeutic agents. Alternatively, a compound of the invention may beadministered to a patient in need thereof in combination with theadministration of one or more other therapeutic regimens (e.g. Gleevecor other kinase inhibitors, interferon, bone marrow transplant, farnesyltransferase inhibitors, bisphosphonates, thalidomide, cancer vaccines,hormonal therapy, antibodies, radiation, etc). For example, additionaltherapeutic agents for conjoint administration or inclusion in apharmaceutical composition with a compound of this invention may beanother one or more anticancer agents.

As described herein, the compositions of the present invention comprisea compound of the invention together with a pharmaceutically acceptablecarrier, which, as used herein, includes any and all solvents, diluents,or other vehicle, dispersion or suspension aids, surface active agents,isotonic agents, thickening or emulsifying agents, preservatives, solidbinders, lubricants and the like, as suited to the particular dosageform desired. Remington's Pharmaceutical Sciences, Fifteenth Edition, E.W. Martin (Mack Publishing Co., Easton, Pa., 1975) discloses variouscarriers used in formulating pharmaceutical compositions and knowntechniques for the preparation thereof. Except insofar as anyconventional carrier medium is incompatible with the compounds of theinvention, such as by producing any undesirable biological effect orotherwise interacting in a deleterious manner with any othercomponent(s) of the pharmaceutical composition, its use is contemplatedto be within the scope of this invention. Some examples of materialswhich can serve as pharmaceutically acceptable carriers include, but arenot limited to, sugars such as lactose, glucose and sucrose; starchessuch as corn starch and potato starch; cellulose and its derivativessuch as sodium carboxymethyl cellulose, ethyl cellulose and celluloseacetate; powdered tragacanth; malt; gelatin; talc; excipients such ascocoa butter and suppository waxes; oils such as peanut oil, cottonseedoil; safflower oil; sesame oil; olive oil; corn oil and soybean oil;glycols; such a propylene glycol; esters such as ethyl oleate and ethyllaurate; agar; buffering agents such as magnesium hydroxide and aluminumhydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer'ssolution; ethyl alcohol, and phosphate buffer solutions, as well asother non-toxic compatible lubricants such as sodium lauryl sulfate andmagnesium stearate, as well as coloring agents, releasing agents,coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the composition.

6. Formulations

This invention also encompasses a class of compositions comprising theactive compounds of this invention in association with one or morepharmaceutically-acceptable carriers and/or diluents and/or adjuvants(collectively referred to herein as “carrier” materials) and, ifdesired, other active ingredients.

In certain embodiments, the invention provides a pharmaceuticalformulation for treating cancer, in particular the cancers describedherein, comprising a compound of the present invention or apharmaceutically acceptable salt thereof together with apharmaceutically acceptable carrier.

In certain embodiments, the invention provides a pharmaceuticalformulation for treating a cancer selected from the group consisting ofcolorectal cancer, mantel cell lymphoma, breast cancer (includingER⁺HER2⁻ advanced or metastatic or recurrent breast cancer in an adultwoman, or a postmenopausal woman), glioblastoma, acute myeloid leukemiaand lung cancer, especially NSCLC, comprising a compound of the presentinvention or a pharmaceutically acceptable salt thereof together with apharmaceutically acceptable carrier.

In certain embodiments, the invention provides a pharmaceuticalformulation for treating glioblastoma or astrocytoma, comprising acompound of the invention and temozolomide, together with apharmaceutically acceptable carrier.

In certain embodiments, the invention also provides a pharmaceuticalformulation, comprising a compound of the invention or apharmaceutically acceptable salt thereof and temozolomide, together witha pharmaceutically acceptable carrier, diluent, or excipient.

In certain embodiments, the invention provides a pharmaceuticalformulation for treating NSCLC, pancreatic cancer, ovarian cancer ormetastatic breast cancer (including ER⁺HER2⁻ advanced or metastatic orrecurrent breast cancer in an adult woman, or a postmenopausal woman),comprising a compound of the invention and gemcitabine HCl, togetherwith a pharmaceutically acceptable carrier.

In certain embodiments, the invention also provides a pharmaceuticalformulation, comprising a compound of the invention or apharmaceutically acceptable salt thereof and gemcitabine HCl, togetherwith a pharmaceutically acceptable carrier, diluent, or excipient.

The active compounds of the present invention may be administered by anysuitable route, preferably in the form of a pharmaceutical compositionadapted to such a route, and in a dose effective for the treatmentintended. The compounds and compositions of the present invention may,for example, be administered orally, mucosally, topically, rectally,pulmonarily such as by inhalation spray, or parentally includingintravascularly, intravenously, intraperitoneally, subcutaneously,intramuscularly, intrasternally and infusion techniques, in dosage unitformulations containing conventional pharmaceutically acceptablecarriers, adjuvants, and vehicles.

The pharmaceutically active compounds of this invention can be processedin accordance with conventional methods of pharmacy to produce medicinalagents for administration to patients, including humans and othermammals.

For oral administration, the pharmaceutical composition may be in theform of, for example, a tablet, capsule, suspension or liquid. Thepharmaceutical composition is preferably made in the form of a dosageunit containing a particular amount of the active ingredient.

Examples of such dosage units are tablets or capsules. For example, asuitable daily dose for a human or other mammal may vary depending onthe condition of the patient and other factors, but, once again, can bedetermined using routine methods.

The amount of compounds which are administered and the dosage regimenfor treating a disease condition with the compounds and/or compositionsof this invention depends on a variety of factors, including the age,weight, sex and medical condition of the subject, the type of disease,the severity of the disease, the route and frequency of administration,and the particular compound employed. Thus, the dosage regimen may varywidely, but can be determined routinely using standard methods. Asmentioned previously, the daily dose can be given in one administrationor may be divided between 2, 3, 4 or more administrations.

For therapeutic purposes, the active compounds of this invention areordinarily combined with one or more adjuvants, excipients or carriersappropriate to the indicated route of administration. If administeredper os, the compounds may be admixed with lactose, sucrose, starchpowder, cellulose esters of alkanoic acids, cellulose alkyl esters,talc, stearic acid, magnesium stearate, magnesium oxide, sodium andcalcium salts of phosphoric and sulfuric acids, gelatin, acacia gum,sodium alginate, polyvinylpyrrolidone, and/or polyvinyl alcohol, andthen tableted or encapsulated for convenient administration. Suchcapsules or tablets may contain a controlled-release formulation as maybe provided in a dispersion of active compound in hydroxypropylmethylcellulose.

In the case of skin conditions, it may be preferable to apply a topicalpreparation of compounds of this invention to the affected area two tofour times a day. Formulations suitable for topical administrationinclude liquid or semi-liquid preparations suitable for penetrationthrough the skin (e.g., liniments, lotions, ointments, creams, orpastes) and drops suitable for administration to the eye, ear, or nose.For topical administration, the active ingredient may comprise from0.001% to 10% w/w, e.g., from 1% to 2% by weight of the formulation,although it may comprise as much as 10% w/w, but preferably not morethan 5% w/w, and more preferably from 0.1% to 1% of the formulation.

The compounds of this invention can also be administered by atransdermal device. Preferably transdermal administration will beaccomplished using a patch either of the reservoir and porous membranetype or of a solid matrix variety. In either case, the active agent isdelivered—continuously from the reservoir or microcapsules through amembrane into the active agent permeable adhesive, which is in contactwith the skin or mucosa of the recipient. If the active agent isabsorbed through the skin, a controlled and predetermined flow of theactive agent is administered to the recipient. In the case ofmicrocapsules, the encapsulating agent may also function as themembrane. The oily phase of the emulsions of this invention may beconstituted from known ingredients in a known manner.

While the phase may comprise merely an emulsifier, it may comprise amixture of at least one emulsifier with a fat or an oil or with both afat and an oil. Preferably, a hydrophilic emulsifier is includedtogether with a lipophilic emulsifier which acts as a stabilizer. It isalso preferred to include both an oil and a fat. Together, theemulsifier(s) with or without stabilizer(s) make-up the so-calledemulsifying wax, and the wax together with the oil and fat make up theso-called emulsifying ointment base which forms the oily dispersed phaseof the cream formulations. Emulsifiers and emulsion stabilizers suitablefor use in the formulation of the present invention include Tween 60,Span 80, cetostearyl alcohol, myristyl alcohol, glyceryl monostearate,sodium lauryl sulfate, glyceryl distearate alone or with a wax, or othermaterials well known in the art.

The choice of suitable oils or fats for the formulation is based onachieving the desired cosmetic properties, since the solubility of theactive compound in most oils likely to be used in pharmaceuticalemulsion formulations is very low. Thus, the cream should preferably bea non-greasy, non-staining and washable product with suitableconsistency to avoid leakage from tubes or other containers. Straight orbranched chain, mono- or dibasic alkyl esters such as di-isoadipate,isocetyl stearate, propylene glycol diester of coconut fatty acids,isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate,2-ethylhexyl palmitate or a blend of branched chain esters may be used.These may be used alone or in combination depending on the propertiesrequired.

Alternatively, high melting point lipids such as white soft paraffinand/or liquid paraffin or other mineral oils can be used.

Formulations suitable for topical administration to the eye also includeeye drops wherein the active ingredients are dissolved or suspended insuitable carrier, especially an aqueous solvent for the activeingredients.

The active ingredients are preferably present in such formulations in aconcentration of 0.5 to 20%, advantageously 0.5 to 10% and particularlyabout 1.5% w/w.

Formulations for parenteral administration may be in the form of aqueousor non-aqueous isotonic sterile injection solutions or suspensions.These solutions and suspensions may be prepared from sterile powders orgranules using one or more of the carriers or diluents mentioned for usein the formulations for oral administration or by using other suitabledispersing or wetting agents and suspending agents. The compounds may bedissolved in water, polyethylene glycol, propylene glycol, ethanol, cornoil, cottonseed oil, peanut oil, sesame oil, benzyl alcohol, sodiumchloride, tragacanth gum, and/or various buffers. Other adjuvants andmodes of administration are well and widely known in the pharmaceuticalart. The active ingredient may also be administered by injection as acomposition with suitable carriers including saline, dextrose, or water,or with cyclodextrin (i.e. Captisol), cosolvent solubilization (i.e.propylene glycol) or micellar solubilization (i.e. Tween 80).

The sterile injectable preparation may also be a sterile injectablesolution or suspension in a non-toxic parenterally acceptable diluent orsolvent, for example as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that may be employed are water,Ringer's solution, and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose any bland fixed oil may be employed,including synthetic mono- or diglycerides. In addition, fatty acids suchas oleic acid find use in the preparation of injectables.

For pulmonary administration, the pharmaceutical composition may beadministered in the form of an aerosol or with an inhaler including drypowder aerosol.

Suppositories for rectal administration of the drug can be prepared bymixing the drug with a suitable nonirritating excipient such as cocoabutter and polyethylene glycols that are solid at ordinary temperaturesbut liquid at the rectal temperature and will therefore melt in therectum and release the drug.

The pharmaceutical compositions may be subjected to conventionalpharmaceutical operations such as sterilization and/or may containconventional adjuvants, such as preservatives, stabilizers, wettingagents, emulsifiers, buffers etc. Tablets and pills can additionally beprepared with enteric coatings. Such compositions may also compriseadjuvants, such as wetting, sweetening, flavoring, and perfuming agents.Pharmaceutical compositions of this invention comprise a compound of theformulas described herein or a pharmaceutically acceptable salt thereof;an additional agent selected from a kinase inhibitory agent (smallmolecule, polypeptide, antibody, etc.), an immunosuppressant, ananticancer agent, an anti-viral agent, antiinflammatory agent,antifungal agent, antibiotic, or an anti-vascular hyperproliferationcompound; and any pharmaceutically acceptable carrier, adjuvant orvehicle.

Alternate compositions of this invention comprise a compound of theformulae described herein or a pharmaceutically acceptable salt thereof;and a pharmaceutically acceptable carrier, adjuvant or vehicle. Suchcompositions may optionally comprise one or more additional therapeuticagents, including, for example, kinase inhibitory agents (smallmolecule, polypeptide, antibody, etc.), immunosuppressants, anti-canceragents, anti-viral agents, antiinflammatory agents, antifungal agents,antibiotics, or anti-vascular hyperproliferation compounds.

The term “pharmaceutically acceptable carrier or adjuvant” refers to acarrier or adjuvant that may be administered to a patient, together witha compound of this invention, and which does not destroy thepharmacological activity thereof and is nontoxic when administered indoses sufficient to deliver a therapeutic amount of the compound.Pharmaceutically acceptable carriers, adjuvants and vehicles that may beused in the pharmaceutical compositions of this invention include, butare not limited to, ion exchangers, alumina, aluminum stearate,lecithin, selfemulsifying drug delivery systems (SEDDS) such asd-atocopherol polyethyleneglycol 1000 succinate, surfactants used inpharmaceutical dosage forms such as Tweens or other similar polymericdelivery matrices, serum proteins, such as human serum albumin, buffersubstances such as phosphates, glycine, sorbic acid, potassium sorbate,partial glyceride mixtures of saturated vegetable fatty acids, water,salts or electrolytes, such as protamine sulfate, disodium hydrogenphosphate, potassium hydrogen phosphate, sodium chloride, zinc salts,colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone,cellulose-based substances, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat. Cyclodextrins such as u-, P-, and y-cyclodextrin, orchemically modified derivatives such as hydroxyalkylcyclodextrins,including 2 and 3-hydroxypropyl-cyclodextrins, or other solubilizedderivatives may also be advantageously used to enhance delivery ofcompounds of the formulae described herein.

The pharmaceutical compositions may be orally administered in any orallyacceptable dosage form including, but not limited to, capsules, tablets,emulsions and aqueous suspensions, dispersions and solutions. In thecase of tablets for oral use, carriers which are commonly used includelactose and corn starch. Lubricating agents, such as magnesium stearate,are also typically added. For oral administration in a capsule form,useful diluents include lactose and dried corn starch. When aqueoussuspensions and/or emulsions are administered orally, the activeingredient may be suspended or dissolved in an oily phase is combinedwith emulsifying and/or suspending agents.

If desired, certain sweetening, flavoring and/or coloring agents may beadded. The pharmaceutical compositions may comprise formulationsutilizing liposome or microencapsulation techniques, various examples ofwhich are known in the art.

The pharmaceutical compositions may be administered by nasal aerosol orinhalation. Such compositions are prepared according to techniques wellknown in the art of pharmaceutical formulation and may be prepared assolutions in saline, employing benzyl alcohol or other suitablepreservatives, absorption promoters to enhance bioavailability,fluorocarbons, and/or other solubilizing or dispersing agents, examplesof which are also well known in the art.

7. Treatment Kits

One aspect of the present invention relates to a kit for convenientlyand effectively carrying out the methods or uses in accordance with thepresent invention. In general, the pharmaceutical pack or kit comprisesone or more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Such kits are especiallysuited for the delivery of solid oral forms such as tablets or capsules.Such a kit preferably includes a number of unit dosages, and may alsoinclude a card having the dosages oriented in the order of theirintended use. If desired, a memory aid can be provided, for example inthe form of numbers, letters, or other markings or with a calendarinsert, designating the days in the treatment schedule in which thedosages can be administered. Optionally associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticalproducts, which notice reflects approval by the agency of manufacture,use or sale for human administration.

The following representative examples contain important additionalinformation, exemplification and guidance which can be adapted to thepractice of this invention in its various embodiments and theequivalents thereof. These examples are intended to help illustrate theinvention, and are not intended to, nor should they be construed to,limit its scope. Indeed, various modifications of the invention, andmany further embodiments thereof, in addition to those shown anddescribed herein, will become apparent to those skilled in the art uponreview of this document, including the examples which follow and thereferences to the scientific and patent literature cited herein.

The contents of the cited references are incorporated herein byreference to help illustrate the state of the art.

In addition, for purposes of this invention, the chemical elements areidentified in accordance with the Periodic Table of the Elements, CASversion, Handbook of Chemistry and Physics, 75^(th) Ed., inside cover.Additionally, general principles of organic chemistry, as well asspecific functional moieties and reactivity, are described in “OrganicChemistry,” Thomas Sorrell, University Science Books, Sausalito: 1999,and “Organic Chemistry,” Morrison & Boyd (3d Ed), the entire contents ofboth of which are incorporated herein by reference.

8. Synthesis Schemes

The compounds of Formula I can be prepared by one of ordinary skill inthe art following art recognized techniques and procedures. Morespecifically, compounds of Formula I can be prepared as set forth in theschemes, methods, and examples set forth below. It will be recognized byone of skill in the art that the individual steps in the followingschemes may be varied to provide the compounds of Formula I. Thereagents and starting materials are readily available to one of ordinaryskill in the art. All substituents, unless otherwise specified, are aspreviously defined.

EXAMPLES Synthetic Examples Equipment Description

¹H NMR spectra were recorded on a Bruker Ascend 400 spectrometer.Chemical shifts are expressed in parts per million (ppm, S units).Coupling constants are in units of hertz (Hz). Splitting patternsdescribe apparent multiplicities and are designated as s (singlet), d(doublet), t (triplet), q (quartet), quint (quintet), m (multiplet), br(broad).

The analytical low-resolution mass spectra (MS) were recorded on WatersACQUITY UPLC with SQ Detectors using a Waters CORTECS C18+, 2.7 μm4.6×30 mm using a gradient elution method.

-   -   Solvent A: 0.1% formic acid (FA) in water    -   Solvent B: 0.1% FA in acetonitrile    -   5% ACN to 95% ACN in 1.0 min, hold 1.0 min,    -   Total 2.5 min; Flow rate: 1.8 mL/min; Column Temp 40 degree.

INTERMEDIATE Intermediate 1

Step 1

To a solution of 4-benzyloxypyridine (185 mg, 998 μmol) in DCM (10 mL)was added amino 2,4,6-trimethylbenzenesulfonate (236 mg, 1.1 mmol) at25° C. The reaction mixture was stirred at 25° C. for 14 h. The mixturewas concentrated under reduced pressure to get crude desired product(400 mg) as colorless oil. LC-MS: m/z 202 [M+H]⁺.

Step 2

To a solution of above product (187 mg, 487 μmol) in DMF (10 mL) wasadded Cs₂CO₃ (192 mg, 1.4 mmol) and but-3-yn-2-one (94 mg, 1.4 mmol).The reaction mixture was stirred at 25° C. for 12 h. The reactionmixture was quenched with water (50 mL) and extracted with DCM (2×25mL). The organic layer was washed with brine (20 mL), dried overanhydrous Na₂SO₄ and concentrated under reduced pressure. The residuewas purified by column chromatography (eluting with PE/EA=10/1) to getdesired product (90 mg, 35% yield) as yellow solid. LC-MS: m/z 267[M+H]⁺.

Step 3

To a solution of methyl(triphenyl)phosphonium bromide (241 mg, 675 μmol)in THF (10 ml) was added butyllithium (43.3 mg, 675 μmol) dropwise underN₂ at −20° C. The reaction was stirred at −20° C. for 1 h. Then asolution of 1-(5-benzyloxypyrazolo[1,5-a] pyridin-3-yl) ethanone (90 mg,338 μmol) in THF (15 ml) was added dropwise at −20° C. The reactionmixture was stirred at 10° C. for 3 h. The reaction mixture was quenchedwith MeOH (3 ml) and concentrated under reduced pressure. The residuewas purified by prep-HPLC (eluting with PE:EA=1/1) to get crude desiredproduct (41 mg) as yellow solid. LC-MS: m/z 265 [M+H]⁺.

Step 4

To a solution of 5-benzyloxy-3-isopropenyl-pyrazolo[1,5-a] pyridine (600mg, 2.3 mmol) in methanol (50 mL) was added Pd/C (60 mg). The reactionmixture was stirred at 30° C. under H₂ for 48 h. The reaction mixturewas filtrated and concentrated under reduced pressure to get desiredproduct (380 mg) as yellow solid. LC-MS: m/z 177 [M+H]⁺.

Step 5

To a solution of 3-isopropylpyrazolo[1,5-a] pyridin-5-ol (650 mg, 3.7mmol) and DIPEA (410 mg, 4.1 mmol) in DCM (15 mL) was added Tf₂O (1.1 g,4.1 mmol) under N₂ at 0° C. The reaction mixture was stirred at 0° C.for 2 h. The reaction mixture was washed with brine (15 mL) and driedover Na₂SO₄. The organic layer was filtrated, and the filtrate wasconcentrated to get desired product (1.1 g) as colorless oil. LC-MS: m/z309 [M+H]⁺.

Step 6

To a solution of (3-isopropylpyrazolo[1,5-a]pyridin-5-yl)trifluoromethanesulfonate (1.1 g, 3.4 mmol) and4,4,5,5-tetramethyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3,2-dioxaborolane(1.3 g, 5.1 mmol) in dioxane (10 mL) were added Pd(dppf)Cl₂ (249 mg, 340μmol) and KOAc (1.0 g, 10.2 mmol). The reaction mixture was stirred at110° C. for 2 h under N₂. The mixture was filtrated, and the filtratewas concentrated under reduced pressure to obtained crude desiredproduct (950 mg) as dark solid. LC-MS: m/z 287 [M+H]⁺.

Step 7

To a solution of3-isopropyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrazolo[1,5-a]pyridine(950 mg, 3.3 mmol) and 2,4-dichloro-5-fluoro-pyrimidine (665 mg, 4.0mmol) in H₂O (1 mL) and 1,4-dioxane (15 mL) were added Na₂CO₃ (1.2 g, 10mmol) and Pd(dppf)Cl₂ (242 mg, 332 μmol). The mixture was stirred underN₂ at 110° C. for 6 h. The mixture was concentrated under reducedpressure and the residue was purified by flash column chromatography(PE/EA with EA 0-50%) to afford desired product (650 mg, 67% yield) asyellow solid. LC-MS: m/z 291 [M+H]⁺.

Intermediate 2

To a solution of3-isopropyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrazolo[1,5-a]pyridine(3.5 g, 12.2 mmol) and 2,4-dichloropyrimidine (2.7 g, 18.4 mmol) inwater (3 mL) and 1,4-dioxane (60 mL) were added Pd(dppf)Cl₂ (0.9 g, 1.2mmol) and Na₂CO₃ (1.52 g, 14 mmol). The reaction mixture was stirredunder N₂ at 110° C. for 6 h. The mixture was concentrated under reducedpressure and the residue was purified by flash column chromatography (PEwith EA 0-50%) to afford desired product (2.1 g, 62% yield) as yellowsolid. LC-MS: m/z 273 [M+H]⁺.

Intermediate 3

Step 1

To a mixture of 2,6-dichloro-3-nitropyridin-4-amine (1500 g, 7.2 mol)and Iron powder (1933 g, 34.6 mmol) in ethyl alcohol (45 L) and water (3L) was added HCl (1.5 L, 12 M in H₂O) in water (6.5 L) drop-wise at 0°C. in 1 hour, the resulting mixture was stirred at 95° C. for 16 hours.The mixture was cooled to room temperature and then neutralized withsodium hydrogen carbonate (solid) to pH=9. The mixture was filtered andwashed with ethyl acetate (500 mL). The filtrate was concentrated toremove solvent. Then the solution was extracted with ethyl acetate (9L). The combined organic layers were washed with brine (1 L), dried overanhydrous sodium sulfate and filtered. The filtrate was concentrated togive 2,6-dichloropyridine-3,4-diamine (1200 g) as yellow solid.

Step 2

The solution of 2,6-dichloropyridine-3,4-diamine (1200 g, 6.74 mol) intriethoxy methane (3 L) was stirred under nitrogen atmosphere at 140° C.for 28 hours. The reaction was concentrated. Formic acid (1.5 L) wasadded. The resulting mixture was stirred at 120° C. for 2 hours. Thesolution was concentrated to give a residue, which was triturated withpetroleum ether/ethyl acetate (1/1, 400 mL) to give4,6-dichloro-1H-imidazo[4,5-c]pyridine (1360 g) as yellow solid.

Step 3

The mixture of 4,6-dichloro-1H-imidazo[4,5-c]pyridine (1360 g, 5.8 mol),K₂CO₃ (5680 g, 17.4 mol) and 2-iodopropane (3951 g, 23.2 mol) in DMF (5L) was stirred under nitrogen atmosphere at 20° C. for 24 hours. Ethylacetate (40 L) was added to the reaction and the mixture was filtered.The filtrate was concentrated to give a residue, which was purified bysilica gel column (petrol ether/ethyl acetate from 3:1 to 1:1) to givedesired product (710 g) as yellow solid.

Step 4

To the mixture of 4,6-dichloro-1-isopropyl-imidazo[4,5-c] pyridine (250mg, 1.1 mmol) in DMSO (10 mL) was added CsF (510 mg, 3.4 mmol), then themixture stirred at 140° C. for 1.5 hours. The resulting mixture werepoured into water (100 mL) and extracted with EA (3×30 mL). The combinedorganic layers were dried over Na₂SO₄ and filtered. The filtrate wasconcentrated. The residue was purified by flash column (80 g 200-300mesh sic-gel, PE/EA=5/1-2/1) to afford desired product (210 mg, 75%yield) as a cream white solid. LC-MS: m/z 214.1 [M+H]⁺.

Step 5

To a solution of 6-chloro-4-fluoro-1-isopropyl-imidazo[4,5-c] pyridine(30 mg, 140 μmol) and4,4,5,5-tetramethyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3,2-dioxaborolane(35.6 mg, 140 μmol) in dioxane (5 mL) were added potassium acetate (41.3mg, 421 μmol) and cyclopentyl(diphenyl) phosphane dichloropalladium iron(15.4 mg, 21.1 μmol). The mixture was degassed with N₂ and stirred at110° C. for 16 hours. The mixture was filtered through a Celite pad. Thefiltrate was concentrated under reduced pressure to crude desiredproduct (50 mg) as black oil, which was used directly in the next step.LC-MS: m/z 224.2 [M+H]⁺.

Step 6

To a solution of (4-fluoro-1-isopropyl-imidazo[4,5-c] pyridin-6-yl)boronic acid (50 mg, 224 μmol) and 2-chloro-4-iodo-pyrimidine (53.9 mg,224 μmol) in dioxane (3 mL) were added Pd(dppf)Cl₂ (24.6 mg, 33.6 μmol)and KOAc (66 mg, 672 μmol). The mixture was degassed with N₂ and stirredat 110° C. for 16 hours. The mixture was concentrated under reducedpressure. The residue was purified by flash chromatography eluting withethyl acetate in petroleum ether 0-60% to give desired product (30 mg,46% yield) as white solid. LC-MS: m/z) 292.1 [M+H]⁺.

Intermediate 4

Step 1

To a solution of 5-bromo-2-nitro-pyridine (1 g, 4.9 mmol) and1-isopropylpiperazine (631.6 mg, 4.9 mmol) in dioxane (40 mL) was addedtris(dibenzylideneacetone)dipalladium(0) (451.1 mg, 492 μmol),(5-diphenylphosphanyl-9,9-dimethyl-xanthen-4-yl)-diphenyl-phosphane (570mg, 985 μmol) and cesium carbonate (4.8 g, 14.8 mmol). Then the reactionmixture was stirred at 110° C. under N₂ for 3 hr. The reaction mixturewas concentrated under reduced pressure and purified by silica gelchromatography eluting with ethyl acetate in petroleum ether 1-100% toafford desired product (850 mg, 68% yield) as a yellow solid. LC-MS: m/z251.1 [M+H]⁺.

Step 2

To a solution of 1-isopropyl-4-(6-nitro-3-pyridyl)piperazine (850 mg,3.4 mmol) in methanol (30 mL) was added Pd/C (412 mg, 10%). Then thereaction mixture was degassed with H₂ for three time and stirred at 25°C. for 3 hr. The reaction mixture was filtered and then washed withmethanol (20 mL). The combined solvent was concentrated under reducedpressure to give desired product (620 mg, 82% yield) as a brown solid.LC-MS: m/z 221.2 [M+H]⁺.

Intermediate 5

Step 1

To a mixture of piperidine-2,4-dione (2.1 g, 18.5 mmol) andN-methylmethanamine (3.4 g, 74.2 mmol) in DCM (36 mL) and THF (18 mL)was added CH₃COOH (10 mL), the resulting mixture was stirred undernitrogen atmosphere at 25° C. for 3 h. Sodium triacetoxyborohydride (7.8g, 37.1 mmol) was added to this mixture, the resulting mixture wasstirred under nitrogen atmosphere at 25° C. for 12 h. The reaction wasquenched with water (50 mL) and concentrated in vacuo to remove DCM andTHF. The mixture was extracted with DCM (3×100 mL). The organic solutionwas washed with brine (20 mL). The organic phase was dried over Na₂SO₄,filtered and concentrated under reduced pressure to afford desiredproduct (2.6 g), which was used in next step without any purification.LC-MS: m/z 141.2 [M+H]⁺.

Step 2

To a mixture of 4-(dimethylamino)-2,3-dihydro-1H-pyridin-6-one (1.0 g,7.1 mmol) in methanol (15 mL) was added sodium borohydride (539 mg, 14.2mmol), the resulting mixture was stirred under nitrogen atmosphere at25° C. for 12 h. The reaction was quenched with sat. NH₄Cl aqueoussolution (10 mL) and then concentrated in vacuo to remove MeOH. Theaqueous solution was purified by reverse phase column (C18, 40 g)eluting with (MeCN/water (0.1% NH₄OH)=1/10) to give desired product (0.3g, 32% yield) as light yellow solid. LC-MS: m/z 143.2 [M+H]*.

Step 3

To a mixture of 4-(dimethylamino)piperidin-2-one (270 mg, 1.9 mmol),5-iodopyridin-2-amine (1.0 g, 4.7 mmol) and potassium phosphate (1.2 g,5.7 mmol) in dioxane (26 mL) was added(1S,2S)-N1,N2-dimethylcyclohexane-1,2-diamine (162 mg, 1.1 mmol) and CuI(108 mg, 569 μmol), the resulting mixture was stirred under nitrogenatmosphere at 110° C. for 12 h. The reaction was filtered. The filtratewas concentrated in vacuo to give the residue, which was purified byreverse phase column (C18, 20 g) eluting with (MeCN/water (0.1%NH₄OH)=1/10) to give desired product (272 mg, 61% yield) as alight-yellow solid. LC-MS: m/z 235.2 [M+H]⁺.

Synthetic Example 1

To a mixture of 1-(6-amino-3-pyridyl)-4-(dimethylamino)piperidin-2-one(30 mg, 128 μmol) and5-(2-chloropyrimidin-4-yl)-3-isopropyl-pyrazolo[1,5-a]pyridine (38.4 mg,140.8 μmol) in dioxane (5 mL) was added cesium carbonate (125.1 mg,384.1 μmol), tris(dibenzylideneacetone)dipalladium(0) (11.7 mg, 12.8μmol) and RuPhos (11.9 mg, 25.6 μmol). The resulting mixture was stirredunder nitrogen atmosphere at 110° C. for 4 h. The reaction mixture wasextracted with EA (20 mL). The organic phase was washed with water (3×20mL), brine (3×20 mL) and dried over Na₂SO₄. The mixture was concentratedunder reduced pressure and purified by flash column chromatography(DCM/MeOH=10:1) to afford desired product (23.8 mg, 39% yield) as ayellow solid. LC-MS: m/z 471.2 [M+H]⁺.

Synthetic Examples 2 and 3

N-[5-[4-(dimethylamino)-1-piperidyl]-2-pyridyl]-4-(3-isopropylpyrazolo[1,5-a]pyridin-5-yl)pyrimidin-2-amine(210 mg, 459.9 μmol) was chiral separated by SFC with mobile phase(Hexane/EtOH/DEA=60/40/0.1) (wave length: UV 214 nm, Column: CHIRALCELOD-H 5.0 cm I.D.×25 cm L, Flow rate: 60 mL/min) to give syntheticexample 2 (44.2 mg, 21% yield) as a yellow solid (LC-MS: m/z 471.2[M+H]⁺. ee value >99%) and synthetic example 3 (41 mg, 19% yield) as ayellow solid (LC-MS: m/z 471.2 [M+H]⁺. ee value=97%).

Synthetic Example 4

To a solution of 5-(4-isopropylpiperazin-1-yl)pyridin-2-amine (124.6 mg,565 μmol) and6-(2-chloropyrimidin-4-yl)-4-fluoro-1-isopropyl-imidazo[4,5-c]pyridine(150 mg, 514 μmol) in dioxane (15 mL) was added Pd₂(dba)₃ (47.1 mg, 51μmol), RuPhos (47.9 mg, 102 μmol) and Cs₂CO₃ (502.6 mg, 1.5 mmol). Themixture was stirred at 110° C. under N₂ for 3 hr. The reaction mixturewas concentrated under reduced pressure. The residue was purified bysilica gel chromatography eluting with MeOH in DCM 0-10% to afforddesired product (107.2 mg, 43% yield) as a yellow solid. LC-MS: m/z476.2 [M+H]⁺.

Biological Example 1. Assay for Inhibition of CDK4/CyclinD1

The CDK4 enzyme assay for IC₅₀ determination was performed as follows.Microfluidic kinase detection technology (Caliper) was used to monitorthe phosphorylation of peptide substrate by CDK4/CyclinD1. The totalreaction volume was 15 μL containing buffer A (100 mM HEPES (pH 7.5),0.1% BSA, 0.01% Triton X-100, 1 mM DTT, 10 mM MgCl₂, 10 μM SodiumOrthovanadate, 10 μM Beta-Glycerophosphate), 200 μM ATP, 1 nMCDK4/CyclinD1 (Thermofisher, PR8064A), 1 μM FL-34(5-FAM-RRRFRPASPLRGPPK), and the test compound at appropriate dilutionsin DMSO. All components were added to the 384-well plate (Corning,4514), and incubated at Room Temperature for 3 hours. The reaction wasterminated by addition of 15 μL Stop Buffer (180 mM HEPES (pH 7.5), 20mM EDTA, Coating-3 reagent (PerkinElmer, 760050)). The plate was thenloaded on Caliper EZ Reader (EZ Reader II, PerkinElmer, HD-4HYSG2772),and the reaction mixtures including substrate and product were sippedinto the microfluidic chip for separation and detection. The IC₅₀ valuesof the test compound were determined by fitting the inhibition curves by4 parameter sigmoidal dose-response model using the Xlfit5/GraphPadPrism 5 software.

Biological Example 2. Assay for Inhibition of CDK6/CyclinD3

The CDK6 enzyme assay for IC₅₀ determination was performed as follows.Microfluidic kinase detection technology (Caliper) was used to monitorthe phosphorylation of peptide substrate by CDK6/CyclinD3. The totalreaction volume is 15 μL containing buffer A (100 mM HEPES (pH 7.5),0.1% BSA, 0.01% Triton X-100, 1 mM DTT, 10 mM MgCl₂, 10 μM SodiumOrthovanadate, 10 μM Beta-Glycerophosphate), 300 μM ATP, 2 nMCDK6/CyclinD3 (Carna, 04-107), 1 μM FL-34 (5-FAM-RRRFRPASPLRGPPK), andthe test compound at appropriate dilutions in DMSO. All components wereadded to the 384-well plate (Corning, 4514), and incubated at RoomTemperature for 3 hours. The reaction was terminated by addition of 15μL Stop Buffer (180 mM HEPES (pH 7.5), 20 mM EDTA, Coating-3 reagent(PerkinElmer, 760050)). The plate was then loaded on Caliper EZ Reader(EZ Reader II, PerkinElmer, HD-4HYSG2772), and the reaction mixturesincluding substrate and product were sipped into the microfluidic chipfor separation and detection. The IC₅₀ values of the test compound weredetermined by fitting the inhibition curves by 4 parameter sigmoidaldose-response model using the Xlfit5/GraphPad Prism 5 software.

Biological Example 3. Assay for Inhibition of CDK2/CyclinE1

The CDK2 enzyme assay for IC₅₀ determination was performed as follows.Microfluidic kinase detection technology (Caliper) was used to monitorthe phosphorylation of peptide substrate by CDK2/CyclinE1. The totalreaction volume was 15 μL containing buffer A (100 mM HEPES (pH 7.5),0.1% BSA, 0.01% Triton X-100, 1 mM DTT, 10 mM MgCl₂, 10 μM SodiumOrthovanadate, 10 μM Beta-Glycerophosphate), 100 μM ATP, 5 nMCDK2/CyclinE1 (SignalChem, C29-18G), 5 μM FL-18 (5-FAM-QSPKKG-NH2), andthe test compound at appropriate dilutions in DMSO. All components wereadded to the 384-well plate (Corning, 4514), and incubate at RoomTemperature for 3 hours. The reaction was terminated by addition of 15μL Stop Buffer (180 mM HEPES (pH 7.5), 20 mM EDTA, Coating-3 reagent(PerkinElmer, 760050)). The plate was loaded on Caliper EZ Reader (EZReader II, PerkinElmer, HD-4HYSG2772), and the reaction mixturesincluding substrate and product were sipped into the microfluidic chipfor separation and detection. The IC₅₀ values of the test compound weredetermined by fitting the inhibition curves by 4 parameter sigmoidaldose-response model using the Xlfit5/GraphPad Prism 5 software.

The IC₅₀ values of each exemplified compound against CDK2, CDK4 and CDK6are provided in the synthetic examples below. The IC₅₀ values areindicated as “A,” “B,” “C,” and “D,” for values less than or equal to 10nM; less than or equal to 100 nM; less than or equal to 1 μM; andgreater than 1 μM, respectively.

Biological Example 4. Anti-Proliferation Assay in T47D Cell

T47D is a human breast cancer cell line commonly used in biomedicalresearch involving the hormonal expression of cancer cells. T47D cellsare distinct from other human breast cancer cells in that theirprogesterone receptors (PR) are not regulated by estradiol, a hormonethat is abundant within the cells themselves. T47D cells have beenemployed in studies of the effects of progesterone on breast cancer andthe corresponding transcriptional regulation caused by introduced drugs.The cells have been noted to be extremely resistant to estrogens andantiestrogens.

T47D breast cancer cells from American Type Culture Collection (ATCC,HTB-133) were plated at 3000 cells/well in 96-well plates, and wereincubated in RPMI 1640 medium (Gibco, 31800105) with 10% Fetal BovineSerum (FBS, Biowest, FB-1058) at 37° C., 5% CO₂. After overnightincubation, baseline values were measured of the samples from one plateusing Cyquant reagent (Invitrogen, C35011) following manufacturer'srecommendations. Cells were incubated with the detection reagent for 1hour at 37° C., and then the fluorescence was measured with excitationat 485 nm and emission at 535 nm using Spectra Max M5 (MolecularDevices, HD-4HYSG3196). Other plates were dosed with compounds at aten-point dose concentration from 10 μM to 0.51 nM in a 3-fold dilutionscheme. On day 6 after compound addition, Cyquant reagent was added andthe fluorescence was measured using Spectra Max M5. The IC₅₀ values ofthe test compound's anti-proliferation activity were determined from thebaseline subtracted viability readout curve using Xlfit5/GraphPad Prism5 software.

Biological Example 5. Inhibition of Phosphorylation of RetinoblastomaProtein (pRb) in T47D Cell

T47D breast cancer cells from American Type Culture Collection (ATCC,HTB-133) were plated at 40,000 cells/well in 96-well plates, and wereincubated in RPMI 1640 medium (Gibco, 31800105) with 10% Fetal BovineSerum (FBS, Biowest, FB-1058). Cells were then allowed to adhereovernight at 37° C., 5% CO₂. The following day, compounds were titratedin a 3-fold dilution scheme, and the highest compound concentrationtested was 10 PM. After 24 hours incubation with compounds, cells werelysed in ice-cold lysis buffer containing phosphatase inhibitor cocktailand 1 mM PMSF. Cell lysates (50 μL/well) were then transferred to ELISAplates (pRb Ser807/811 ELISA kit, Cell Signaling, 13152 or pRb Ser780ELISA kit, Cell Signaling, 13016)). Plates were incubated overnight at4° C., with constant slow speed shaking. After incubation, plates werewashed following manufacturer's recommendations, and then 100 μLreconstituted detection antibody was added to each well and incubatedfor 1 hour at 37° C. After incubation, plates were washed, and then 100μL reconstituted HRP-linked secondary antibody was added to each welland incubated for 30 mins at 37° C. After incubation, plates werewashed. Then, 100 μL TMB substrate was added to each well and incubatedfor 10 min at 37° C. or 30 min at 25° C. Finally, 100 μL of STOPsolution was added to each well and mixed gently for a few seconds.Plates were read on the Envision plate reader (PerkinElmer, 2104-0010)using the 96-well luminescence mode. IC₅₀ values were calculated using 4parameter sigmoidal dose-response model of Xlfit5/GraphPad Prism 5software.

The cellular data obtained from biological examples 4 and 5 are listedin the Table A below. The IC₅₀ values are indicated as “++++”, forvalues less than or equal to 100 nM; “+++”, for values less than orequal to 500 nM; “++”, for values less than or equal to 1 μM; and “+”,for values greater than 1 μM, respectively.

TABLE A Phospho Synthetic CDK4 CDK6 CDK2 T47D T47D Ser807 Example IC₅₀IC₅₀ IC₅₀ IC₅₀ IC₅₀ 1 A A B ++++ ++++ 2 A B B ++++ ++++ 3 A A A ++++++++ 4 A B C ++++ ++++

1. (canceled)
 2. A compound represented by the following structuralformula:

or a pharmaceutically acceptable salt or a stereoisomer thereof.
 3. Apharmaceutical composition comprising an effective amount of thecompound of claim 2 or a pharmaceutically acceptable salt thereof, and apharmaceutically acceptable carrier.
 4. A method of treating a cancercomprising administering to a subject in need thereof an effectiveamount of a compound of claim 2, or a pharmaceutically acceptable saltthereof, wherein the cancer is a carcinoma of the bladder, breast,colon, kidney, epidermis, liver, lung, oesophagus, gall bladder, ovary,pancreas, stomach, cervix, thyroid, nose, head and neck, prostate, orskin; a hematopoietic tumor of lymphoid lineage; a hematopoietic tumorof myeloid lineage; thyroid follicular cancer; a tumor of mesenchymalorigin; a tumor of the central or peripheral nervous system; melanoma;seminoma; teratocarcinoma; osteosarcoma; xeroderma pigmentosum;keratoctanthoma; thyroid follicular cancer; or Kaposi's sarcoma.
 5. Amethod of inhibiting activity of a cyclin-dependent kinase (CDK) in asubject, said method comprising administering to the subject aneffective amount of a compound of claim 2, or a pharmaceuticallyacceptable salt thereof.
 6. The method of claim 5, wherein the subjecthas a cancer.
 7. (canceled)
 8. The method according to claim 6, whereinthe cancer is pRb⁺ breast cancer, or hormone receptor (HR)-positiveHER2/neu-negative cancer.
 9. The method of claim 8, wherein the canceris advanced or metastatic or recurrent breast cancer.
 10. The method ofclaim 9, wherein the breast cancer is in an adult woman, or apostmenopausal woman.
 11. The method of claim 8, further comprisingadministering a second agent selected from: an aromatase inhibitor, aSelective Estrogen Receptor Modulator (SERM), a pure antiestrogen withno estrogen agonist activity, a compound that temporarily suppressesovarian function, a compound that inhibits CCYP3A4, or a monoclonalantibody or an antigen-binding fragment thereof against IGF-1/IGF-2. 12.The method of claim 4, further comprising administering an immunecheckpoint inhibitor, a receptor Tyr kinase inhibitor, and/or anantagonist of hormone receptor.
 13. The method of claim 6, wherein thecancer is selected from a carcinoma of the bladder, breast, colon,kidney, epidermis, liver, lung, oesophagus, gall bladder, ovary,pancreas, stomach, cervix, thyroid, nose, head and neck, prostate, orskin; a hematopoietic tumor of lymphoid lineage; a hematopoietic tumorof myeloid lineage; thyroid follicular cancer; a tumor of mesenchymalorigin; a tumor of the central or peripheral nervous system; melanoma;seminoma; teratocarcinoma; osteosarcoma; xeroderma pigmentosum;keratoctanthoma; thyroid follicular cancer; or Kaposi's sarcoma.
 14. Themethod of claim 13, wherein the hematopoietic tumor of lymphoid lineageis leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia,B-cell lymphoma, T-cell lymphoma, multiple myeloma, Hodgkin's lymphoma,non-Hodgkin's lymphoma, hairy cell lymphoma, or Burkett's lymphoma. 15.The method of claim 8, wherein the hormone receptor (HR) is estrogenreceptor positive (ER⁺), progesterone receptor positive (PR⁺), orER⁺PR⁺.
 16. The method of claim 11, wherein the compound thattemporarily suppresses ovarian function is a gonadotropin-releasinghormone (GnRH) agonist or a luteinizing hormone-releasing hormone(LH-RH) agonist.
 17. The method of claim 12, wherein the immunecheckpoint inhibitor is a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4inhibitor.
 18. The method of claim 12, wherein the hormone receptor isan estrogen receptor.